Supporting data for "Enhanced lineage reprogramming towards induced neural stem cells with re-engineered SOX17"

Forced expression of transcription factors (TFs) can program cellular fate changes. The direct transdifferentiation of induced neural stem cells (iNSCs) from somatic cells with defined TFs has been reported in several studies. iNSCs have great potentials in regenerative medicine and as authentic models for age-associated neurological diseases. Compared to cells derived from induced pluripotent stem cells (iPSCs), iNSCs could be a shortcut for the generation of cell models, avoid oncogenic cell states and circumvent the rejuvenation confounding authentic models of age-associated diseases. However, low efficiency, slow speed, poor reproducibility and an unresolved mechanism of iNSC reprogramming impede their routine applications in research and for clinical studies. <br>In this study, to enhance and dissect iNSC generation, we aimed to identify artificially evolved and enhanced TFs (eTFs) to boost iNSC reprogramming based on a Brn4, Sox2, Klf4 and c-Myc (BSKM) cocktail. We identified mutant variants of the endoderm factor Sox17 that can effectively produce iNSCs whereas wild-type Sox2 and Sox17 fail. An engineered eSox17 with three point mutations can drive efficient and rapid generation of iNSCs from fetal, adult and old mouse fibroblasts. These iNSCs are long-term self-renewal, express neural stem cell markers and are capable of differentiating into neurons, astrocytes and oligodendrocytes. <br>By scaling down the number of TFs in reprogramming cocktail, we found exogenous Brn4 and c-Myc factors are dispensable. eSox17 and Klf4 are necessary and sufficient to generate iNSCs. This two-factor cocktail drives direct reprogramming to neural lineage without passing a pluripotent state, demonstrated by the lack of pluripotency gene activation using an Oct4-GFP reporter and Nanog-CreER lineage tracing system. In addition, metabolic assays revealed that Klf4 induces glycolysis essential for iNSC reprogramming. <br>To uncover the molecular mechanism underlying the unique ability of eSox17 to generate iNSCs, we investigated transcriptional dynamic and chromatin accessibility during neural reprogramming. Global gene expression and chromatin opening are similar at the early stage of reprogramming with different Sox factors.Compared with Sox2 and Sox17, eSox17 differentially targets genomic loci with canonical Sox:Oct DNA elements and activates a small subset of genes to mediate cell fate change during neural reprogramming. Importantly, exogenous TF silencing is required to mature reprogramming cells into iNSCs at the late stage. <br>In summary, this study shows eSox17 enables robust reprogramming of somatic cells into iNSCs. The translation of these findings to human iNSC generation could lead to readily available, authentic and personalized cell models for neurodegenerative diseases that bypass the shortcomings associated with iPSC-based strategies. The application of eTFs to generate desired cell types could provide promising cell sources for research and regenerative medicine.

强制表达转录因子（transcription factors, TFs）可介导细胞命运重编程。已有多项研究报道，利用特定转录因子可将体细胞直接转分化为诱导神经干细胞（induced neural stem cells, iNSCs）。iNSCs在再生医学领域具有巨大应用潜力，同时可作为年龄相关性神经疾病的可靠研究模型。相较于诱导多能干细胞（induced pluripotent stem cells, iPSCs）来源的细胞，iNSCs可作为构建细胞模型的快捷路径，能够规避致癌细胞状态，且可避免因重编程返老还童现象对年龄相关性疾病模型真实性造成的干扰。然而，iNSCs重编程存在效率低下、周期漫长、重复性差且机制未明等问题，阻碍了其在基础研究与临床研究中的常规应用。
本研究为优化并解析iNSCs的生成过程，旨在基于Brn4、Sox2、Klf4及c-Myc（BSKM）组合因子，筛选经人工进化增强的转录因子（enhanced TFs, eTFs）以提升iNSCs重编程效率。我们发现，内胚层因子Sox17的突变体可有效诱导生成iNSCs，而野生型Sox2与Sox17则无此效果。携带3个点突变的工程化eSox17可高效、快速地从胎鼠、成年小鼠及老年小鼠的成纤维细胞中诱导生成iNSCs。此类iNSCs可长期自我更新，表达神经干细胞标志物，并能够分化为神经元、星形胶质细胞与少突胶质细胞。
通过精简重编程组合中的转录因子数量，我们发现外源Brn4与c-Myc因子并非必需。仅eSox17与Klf4即可满足iNSCs的诱导生成需求。该双因子组合可直接将体细胞重编程为神经谱系细胞，无需经历多能性状态：通过Oct4-GFP报告基因与Nanog-CreER谱系示踪系统检测，未观察到多能性基因的激活。此外，代谢分析显示，Klf4可诱导iNSCs重编程过程中必需的糖酵解反应。
为揭示eSox17诱导iNSCs生成的独特分子机制，我们探究了神经重编程过程中的转录动态变化与染色质可及性。不同Sox因子介导的重编程早期阶段，全局基因表达与染色质开放状态相似。相较于Sox2与Sox17，eSox17可差异性靶向结合携带经典Sox:Oct DNA元件的基因组位点，并激活少量基因以介导神经重编程过程中的细胞命运转变。尤为重要的是，在重编程后期需对外源转录因子进行沉默，才能将重编程细胞成熟为iNSCs。
综上，本研究证实eSox17可实现体细胞向iNSCs的高效重编程。将该研究成果转化至人类iNSCs的诱导生成，有望为神经退行性疾病提供便捷易得、真实可靠且个性化的细胞模型，规避基于iPSCs的技术方案存在的缺陷。利用增强型转录因子诱导生成目标细胞类型，可为基础研究与再生医学提供极具前景的细胞来源。